The objective of ARISYS is to overcome current limitations for antibody production that are inherent to the extant immune system of vertebrates and therefore generate antibodies á la carte in a short period of time and in a cheap and easy manner. This will be done by creating an all-in-one artificial/synthetic platform based exclusively on prokaryotic parts, devices and modules. To this end, ARISYS will exploit design concepts, construction hierarchies and standardization notions that stem from contemporary Synthetic Biology for the assembly and validation of such a complex artificial biological system. This all-bacterial immune-like system will not only simplify and make affordable the manipulations necessary for antibody generation, but will also permit the application of the resulting binders by themselves or displayed on bacterial cells to biotechnological challenges well beyond therapeutic and health-related uses. The ongoing work involves the assembly and validation of autonomous functional modules for [i] displaying antibody/affibody (AB) scaffolds attached to the surface of bacterial cells, [ii] conditional diversification of target-binding sequences of the ABs, [iii] contact-dependent activation of gene expression, [iv] reversible bi-stable switches, and [v] clonal selection and amplification of improved binders. These modules composed of stand-alone parts and bearing well defined input/output functions, are being assembled in the streamlined genomic chassis of the non-pathogenic bacteria Escherichia coli and Pseudomonas putida. The resulting molecular network will make the ABs expressed and displayed on the cell surface to proceed spontaneously (or at the user's decision) through subsequent cycles of affinity and specificity maturation towards antigens or other targets presented to the bacterial population. In this way, a single, easy-to handle (albeit conceptually complex and heavily engineered) strain will govern all operations that are typically scattered in a multitude of separate methods and apparatuses for AB production. Our vision is one in which the time between identification of a target antigen (or mixtures of antigens) and the generation of large amounts of a neutralizing antibodies can be shortened from months to days. This involves the design of the biological component of the system but also its optimized performance in an actual, simple-to-use mili-fluidic device where all operations involved in the process can be done in a continuous liquid flow.

During the first stage of the Project most of the genetic devices that are necessary for the assembly of the integrated AB-producing platform have been developed, tested and parameterized. The current state of affairs is that of adjustment of such parameters within ranges in which the whole assembly of components can function in a coherent manner. Many of the ongoing efforts are focused on the components that have to make bacteria activate a transcriptional response of a distinct promoter upon physical attachment of the cells to a solid surface. We are also trying and comparing the efficiency of various diversity-generating genetic devices that concentrate the production of DNA sequence variability in specific sites of the AB genes and thus in cognate sites of the corresponding protein. But we are concerned not only with the biological ingredient of the platform: at the same time, we have started to envision how an actual antibody-producing machine could be designed in a user-friendly fashion. In this sense, we expect this Project to deliver not only more fundamental knowledge on AB production, mechanisms of genetic diversification and surface display of heterologous proteins on bacterial cells surfaces, but also a groundbreaking platform to produce large amounts of ABs that is flexible and amenable to use in a plethora of biotechnological directions.